The E2F transcription factor is an important regulator of the G1 to S transition of the cell cycle. E2F activity is rate-limiting for S-phase entry, and the deregulation of E2F is believed to occur in most tumor cells as a result of mutations in the pRB pathway. The vast majority of E2F research has been carried out using mammalian cells. Such studies show that there are many different forms of E2F, that these have a wide variety of transcriptional activities, and that E2F activity can be regulated in many different ways. The immense complexity of mammalian E2F has made it difficult to understand how different components of E2F regulation are integrated together, and it is often difficult to place studies of individual proteins in the larger picture of E2F function. To complement the study of mammalian E2F proteins, we initiated an investigation of E2F in Drosophila. The first two cycles of this grant have provided the basic groundwork needed to use this system. We identified and characterized two E2F homologs, a DP homolog, and two RB family proteins and the genome sequencing efforts revealed that these are the only family members present in flies, dE2F, dDP and RBF1 regulate S-phase entry in vivo in ways that are strikingly analogous to the action of their mammalian counterparts. In the latest cycle of this grant we explored the functional relationships between dE2F1 and dE2F2, and RBF1 and RBF2, and uncovered distinct roles for each of these proteins in E2F-regulated transcription. In the next cycle of this grant we propose to use this system to explore, in detail, three fundamental aspects of E2F/RBF function. Each of these Aims is based on findings made in the previous funding period. Aim 1 will investigate a new class of dE2F2/RBF1 and dE2F2/RBF2-repressed promoters that do not appear to be expressed in cycling cells, but are expressed in developmentally restricted patterns. Aim 2 will investigate the functional overlap between RB family proteins in E2F regulation, and the way that this changes during development. In the previous period we discovered that cells homozygous mutant for both de2fl and de2f2 can proliferate. Aim 3 will use E2F- or DP-deficient cells to determine which aspects of cell cycle regulation require E2F activity. [unreadable] [unreadable]